携带人鞘氨醇激酶及突变体基因重组腺病毒载体的制备及表达

目的制备携带人野生型鞘氨醇激酶基因(SPK^wt)及其突变体(SPK^DN)的腺病毒载体，为研究SPK的生物学功能提供高效基因转移载体。方法将野生型和突变体SPK基因亚克隆至穿梭载体pshuttle-cmv，重组的穿梭质粒经Pme线性化后，电转化E.coli BJ-AD-1感受态，获得带有目的基因的腺病毒重组子质粒；重组子经Pac I线性化后，以脂质体介导转染293包装细胞，获得重组腺病毒载体；分别行PCR鉴定重组腺病毒是否携带目的基因，Western blot鉴定目的基因是否表达，酶活性检测判断野生型基因和突变体基因表达活性。结果重组腺病毒带有目的基因，可以感染内皮细胞并正确表达，酶活性分析表明野生型SPK基因增强SPK活性，突变体SPK基因抑制SPK活性。结论成功构建了携带SPK野生型及其突受体的重组腺病毒，为研究SPK的功能提供了物质基础。

Preparation and identification of recombinant adenoviral vectors containing human wild type SPK and its mutant gene

Objective In order to further understand the bioactivity of sphingosine kinase, high efficient replication defective recombinant adenovirus carrying the wild type SPK gene (SPK WT ) and SPK mutant gene (SPK DN ) were constructed respectively. Methods The SPK genes of interest were subcloned into a shuttle vector pshuttle cmv respectively. Pshuttle cmv SPK WT and Pshuttle cmv SPK DN were linearized by PmeI and transformed respectively into the competent cells of E. coli BJ AD 1 cells which contain adenoviral backbone plasmid pAdEasy 1. The linearized recombinant plasmids were transfected into adenovirus 293 packaging cells respectively,and then recombinant adenoviruses were harvested. We performed the PCR, Western blot and enzyme assays to identify the recombinant adenoviruses. Results The recombinant adenoviruses containing the interest gene and being able to infect ECV 304 cells were obtained. Overexpression of wild type SPK gene in ECV 304 cells increased the endogenous SPK activity, whereas overexpression of mutant SPK (SPK DN ) inhibited intracellular SPK activity. Conclusion Recombinant adenoviral vectors can mediate interesting gene expression in cells.